For neuroblastoma are using four cell lines, two MYCN not-amplified and two MYCN amplified cell line that can be grown in a xenograft model. For RMS we are using ten cell lines, tow with PAX3-FOXO1 fusion genes and two with RAS pathway mutated genes. Among other assays we will use the Incutyte system. For the siRNA screen we will use a druggable genome library of over 6000 genes developed by National Center for Advancing Translational Sciences (NCATS). For the drug screen we will use single agent and combination responses of a panel of 1916 drugs (Mechanism Interrogation Plate (MIPE-v4) Library) also developed by NCATS. The content of this library include 765 FDA approved compounds, 49 of which are approved for cancer therapy, 460 in clinical trials (phase 1, 2 or 3), 149 kinase inhibitors. For 1915 of these compounds, the target or mechanism of action is known. The most promising targets and the appropriate siRNA or drug combination will be further evaluated in the xenograft animal models as outlined above. For Rhabdomyosarcoma (RMS), FGFR4 is a rational target given that it is a key regulator of myogenic differentiation and muscle regeneration after injury; it is expressed in myoblasts, but not in differentiated skeletal muscle. We and others have found that FGFR4 is highly expressed in all RMS, and high expression is a diagnostic and prognostic biomarker. It is a direct target and strongly induced by PAX3-FOXO1, PAX3, and PAX7 and we reported that PAX3-FOXO1 established a super-enhancer at the gene's locus. We have reported that approximately 10% of FN-RMS have activating mutations in FGFR4 and that cells harboring FGFR4 mutations are oncogene addicted and sensitive to pharmacological inhibition by small molecules. Therefore, FGFR4 is a key cell surface tyrosine kinase receptor for RMS biology, growth and survival.we are developing monoclonal antibodies and human scFv binders. The majority detect the human FGFR4 protein by both ELISA and by FACS analysis . To mitigate for potential organ toxicity, we are examining FGFR4 expression levels in normal human organs. We are currently performing extensive RNAseq and immunohistochemistry (IHC) analysis of normal organ and rhabdomyosarcoma tissue arrays. We are testing our scFv binders as potential FGFR4 chimeric antigen receptors (CARs) to generate a second-generation CAR receptor lentiviral construct that contains the CD8 transmembrane region, 41BB and CD3zeta intracellular domains and a human EGFR extracellular domain. This design was chosen because of its efficacy in clinical trials and CAR T cell persistence in patient's peripheral blood for several months after therapy. The truncated EGFR in the CAR construct allows for the measurement of transduced T cells as well as therapeutic targeting of CAR T cells with Cetuximab in clinical trials in case of uncontrolled toxicity. Anti-FGFR4 CART cells could lyse RH30 but not RAJI, a FGFR4 negative Burkitt's lymphoma cell line (data not shown). Work is currently underway to validate FGFR4 CAR T cells in-vivo. If successful we anticipate the development of potent immunotherapeutic biologics and cell based therapies for patients with aggressive RMS. All positive hits will be further screened in a wider panel of NB and RMS Xenografts and PDxs.